Food Bioscience 45 (2022) 101492

Contents lists available at ScienceDirect

Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Potential of fungal thermostable alpha amylase enzyme isolated from Hot

springs of Central Anatolia (Turkey) in wheat bread quality
Arzu Ünal a, Asiye Seis Subaşı b, Semra Malkoç c, İjlal Ocak d, S. Elif Korcan e, Elif Yetilmezer f,
Seyhun Yurdugül g, *, Hülya Yaman h, Turgay Şanal b, Alaettin Keçeli j
a
Iğdır University, Faculty of Agriculture, Department of Agricultural Biotechnology, Iğdır, Turkey
b
Ministry of Agriculture and Forestry Field Crops Central Research Institute, Department of Quality and Technology, Ankara, Turkey
c
Eskişehir Technical University, Faculty of Engineering, Department of Environmental Engineering, Eskişehir, Turkey
d
Afyon Kocatepe University, Faculty of Education, Department of Mathematics and Science Education, Division of Science Education, Afyon, Turkey
e
Uşak University, Vocational School of Health Services, Medical Laboratory Techniques Program, Uşak, Turkey
f
Ministry of Agriculture and Forestry Field Crops Central Research Institute, Department of Biotechnology, Ankara, Turkey
g
Bolu Abant İzzet Baysal University, Faculty of Arts and Science, Department of Biology, Bolu, Turkey
h
Bolu Abant İzzet Baysal University, Vocational School of the Technical Sciences of Bolu, Food Technology Program, Bolu, Turkey
j
Pamukkale University, School of Applied Sciences, Department of Organic Farming Business Management, Denizli, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: Many industrial processes may quickly proceed with biotechnological and enzymatic support. Functional and
Alpha-amylase production facilitator properties of enzymes have provided variable advantages for industry. Therefore, enzyme
Thermal spring utilization has become inevitable for industry and supplied financial advantages in case of the high energy
Quality of bread
requirement process. In the study, the amylase enzymes were isolated from thermal spring sources, and the effect
Commercial enzymes
on bread quality was examined. Firstly, fungal amylases were isolated from thermal spring sources (29–98 ◦ C) in
various places around Turkey. After determining the functional properties of amylase enzymes, the most active
enzyme was used in bread production to examine their effect on bread quality. The maximum alpha-amylase
activity (38.6 U/mg) has been detected in Aspergillus niger G 2–1 isolate. When compared with the commer­
cially available one, native alpha-amylase increased the bread volume. A significant difference (p < 0.01) was
found in the color properties and size of the bread between microbial produced in this study and commercially
produced alpha-amylase. Five ppm alpha-amylase addition showed the optimum bread properties for dough
processing.

1. Introduction from thermal spring reserves is seen as significant due to their

Enzyme utilization for industrial-scale is an increasing trend due to
their several advantages such as milder reaction conditions in produc­
tion, remarkable product properties, lower environmental and physio­
logical toxicity (Chapman et al., 2018). The main enzymes present in the
industry are amylases, cellulases, proteases, and lipases. The microbial
amylases are mainly generated by the genus Bacillus among bacteria
(Sundarram & Murthy, 2014) and Aspergillus in fungi (Mobini-Dehkordi
and Javan, 2013). The catalytic activity of microbial enzymes is higher
and stable than plant and animal isolates, and produced more
economically, produced in a bulk amount in a short time, and no un­
desired side product occurs (Adrio & Demain, 2014). The character­
ization of amylolytic enzyme-producing thermophilic fungi isolation
biotechnological potential as well as serving as a model in thermosta­
bility studies (Leveque et al., 2000; Rana et al., 2013).
These enzymes may either produce by solid-state and submerged
fermentation (Sundarram & Murthy, 2014). Nowadays, many microbial
enzymes are used in the food industry, not only the confectionary, meat,
fruit juice but also including the bakery, such as pectinases, cellulases,
proteases, lipases and amylases. Alpha-amylase is an intensively pro­
duced extracellular enzyme from fungal sources and is the most
preferred enzyme in the bakery industry due to its stability of baking
temperature of bread (Nguyen et al., 2002).
Alpha-amylase enzyme increases the gas holding capacity of the
dough and increases bread volume, shelf-life, bleaches the crumb and
forms its texture, maintains a thin crust and its desired color, retards

* Corresponding author.
E-mail address: yurdugul_s@ibu.edu.tr (S. Yurdugül).

https://doi.org/10.1016/j.fbio.2021.101492
Received 24 July 2021; Received in revised form 26 November 2021; Accepted 27 November 2021
Available online 29 November 2021
2212-4292/© 2021 Elsevier Ltd. All rights reserved.
A. Ünal et al. Food Bioscience 45 (2022) 101492

staling, and prevents crumbling. It keeps the quality of the porous 2.3. Preparation of mediums for amylase production
structure of bread (Keskin, 2003). According to Amigo et al. (2021)
maltogenic α-amylases minimize the staling process in bread. Because of The different starch-containing mediums were used to find out the
the positive effects of the alpha-amylase enzyme on bread, this enzyme most efficient amylase activity. The highest efficiency and activity in
was aimed to be obtained by various fungi from thermal springs. For enzyme production were observed in modified SYEM (Merck) adjusted
determining the fungi species, the thermal springs were screened out for to pH:7 and sterilized at 121 ◦ C for 15 min, according to the following
acquiring high activity alpha-amylase enzyme to utilize in bread pro­ composition: Peptone: 0.5 g/100 mL (Merck, Darmstadt, Germany);
duction and develop high-quality bread in the study. The positive effect Beef extract: 0.15 g/100 mL (Merck, Darmstadt, Germany); Yeast
of enzymes such as the retardation of bread staling, having a high re­ extract: 0.15 g/100 mL (Merck, Darmstadt, Germany); NaCl: 0.5 g
action rate of thermostable enzymes, increasing the solubility of sub­ (Merck, Darmstadt, Germany); Starch (Merck, Darmstadt, Germany): 1
strates, and decreasing the microbial contamination will help produce g/100 mL (Saleem & Mohsen, 2014).
better quality bread. The commercial potential of the alpha-amylase
enzyme was excited that this study has a widespread and potential 2.4. The macroscopic and microscopic characterization of isolates
impact (Osho, 2018).
The macroscopic and microscopic investigation of the isolates were
2. Materials and methods carried out by measuring the colony diameters after incubating them on
Malt Extract Agar (MEA) (Merck, Darmstadt, Germany), Czapek’s Yeast
The fungi were isolated from different thermal springs by sampling Extract Agar (CYA) (Merck, Darmstadt, Germany), and Oatmeal Agar
water and soil, with temperatures ranging from 55 to 90 ◦ C around (OMA) (Merck, Darmstadt, Germany) for a week at 27 ± 2 ◦ C in dark­
Afyon, Eskişehir, Uşak and Ankara provinces in Turkey (Table 1), then ness. X10 and X40 light microscope were used to examine the conidial
macroscopic, microscopic and molecular diagnosis of amylase- head, spore, and phialides biseriate structures of thermotolerant fungal
producing thermophilic and mesophilic fungi were carried out. Ampli­ isolates. The isolates were identified by different mycological taxonomic
fication of the gene encoding alpha amylase by polymerase chain reac­ keys at the species level (Hasenekoğlu, 1991; Barnett & Hunter, 1998).
tion (PCR), deoxyribonucleic acid (DNA) sequence analysis, enzyme
activity and optimization studies were performed in isolates found to 2.5. Culture medium and enzyme activity studies
produce alpha amylase enzyme. The isolates with amylase activity were
grown in pilot scale and the enzyme was obtained in spray dried form. 2.5.1. Enzyme activity determination
The enzyme were used for bread production and quality attributes were Amylase enzyme was produced by shake flask method at a temper­
investigated. ature of 40 ◦ C, pH of 4.5, and at an agitation speed of 175 rpm in the
aerobic medium. Its activity was determined via reducing sugars ob­
tained by the enzymatic hydrolysis of the raw starch by the dinitro
2.1. The pre-enrichment of fungi salicylic acid (DNS, Merck, Darmstadt, Germany) method (Miller,
1959). Amylase activity was defined as the amount of enzyme (one unit)
The method suggested by Rico-Munoz et al. (2015), chap. 22 was converting raw starch into 1 μmol reducing sugar (glucose) formed by
performed for the isolation of fungal species from the water and soil hydrolysis in a minute in standard conditions (Singh et al., 2014).
samples. For pre-enrichment, diluted soil samples and water samples
were placed in Erlenmeyer flasks and were kept in a 40–45 ◦ C water bath 2.6. The molecular characterization of the isolates
for 3 h. The fungal species in samples were grown in Starch Yeast Extract
Agar Medium (SYEM) (Merck, Darmstadt, Germany) in petri dishes, and Genomic DNA extraction was carried out using 100 mL cultures grown
colony counts w e r e enumerated following one week of incubation at in Sabouraud medium (Merck, Darmstadt, Germany) (30 ◦ C, 250 rpm) for
40 ◦ C. 48 h. After transferring the mycelia to liquid nitrogen, it was disrupted and
suspended in 5 mL extraction buffer (100 mM Tris- HCl (Merck, Darmstadt,
2.2. The determination of the presence of amylase enzyme in fungi Germany), 10 mM EDTA (Merck, Darmstadt, Germany) pH = 8 and 1% SDS
(Merck, Darmstadt, Germany), then incubated for 30 min at 65 ◦ C. Potas­
Iodine (Merck, Darmstadt, Germany) is an amylase indicator and the sium Acetate (Merck, Darmstadt, Germany) (2 mL 3 M, pH 4.8) was added,
presence of amylase enzyme in the medium is shown by the zone for­ then it was incubated for an hour at 4.0 ◦ C. After the centrifugation of the
mation around colonies. For this reason, the colonies which show liquid phase for 10 min at 10.000×g, it was extracted by phenol and chlo­
transparent zones when iodine solution was added after incubation were roform. 18 S rDNA and ITS (Internal Transcribed Spacer) 1 region were
evaluated as amylase (+) (Saleem & Mohsen, 2014). identified using fungus-specific primers in PCR amplification, and the
sequential analysis was performed by web-based comparative BLAST
analysis. 18D: 5_-CCTGGTTGA TCCTGCCAGTA-3 and 18 R:5_-
Table 1
GCTTGATCCTTCTGCAGGTT-3_ITSl:5_-TCCGTAGG TGAACCTGCG-3 and
Locations, temperatures, and pH value of thermal spring water.
ITS4:5_TCCTCCGCTTATTGATATG-3_ PCR primers were used in the
Locations Thermal Spring Temperature (◦ C) pH
analysis.
AFYON Gazlıgöl Spa (İhsaniye) 64 6.9
Hüdai Spa (Sandıklı) 68 6.6 2.6.1. Pilot scale enzyme production
Heybeli Spa (Bolvadin) 43 5.9
The fungal alpha-amylase enzyme was produced by the shake flask
Ömer-Gecek Spa (Centrum) 98 7.3
ESKİŞEHİR
Uyuzhamam Spa (Alpu) 29 7.6 method, and the activity of the enzyme was determined with the tech­
Yarıkçı Spa (Mihalıççık) 39 7.5 niques suggested by Shanmughapriya et al. (2010). The enzyme in
Kızılinler Spa (Centrum) 38 7.6 powder form was obtained by precipitation and the spray-drying
Sakarılıca Spa (Mihalgazi) 56 7.6
method of the culture produced in medium explained in Section 2.3.
Çifteler Spa (Çifteler) 48 6.7
Aşağı ve Yukarı Spa (Centrum) 48 6.6
Hasırca Spa (Centrum) 38 5.7 2.7. Bread production and quality analyses
Eskişehir Spa (Centrum) 45 6.7
UŞAK Uşak-Afyon Road 65 7.0 2.7.1. Wheat and flour analyses
ANKARA Haymana Spa 44 6.8
Two wheat cultivars (Tosunbey and Kışla) used for bread making

2
A. Ünal et al.
were obtained from Central Research Institute for Field Crops/Ankara/
Turkey. Hectoliter weight of the samples were measured with the 1-L
container (Seedburo Equipment Company, Chicago, IL, USA) and
given as kg/hL. Thousand kernel weight was determined by using
Tripette& Renaud Numigral II (Villeneuve, France) according to the ISO
Method (No: 520:2010). Pearling index (PI) was determined by grain
pearling equipment (Strong Scott, New Prague, USA) with 20 g sample.
After pearling for 1 min the fine particles were removed by sifting (1.2
mm sieve). The PI was calculated by following formula; PI (%) = 5 × (20
– amount of pearled wheat). The wheat samples were milled by using a
Bühler MLU- 202 Pneumatic Laboratory Mill (Uzwil, Switzerland) ac­
cording to the AACC 26–21 (AACC, 2000).
The moisture and protein content of the samples were determined by
AACC Method 44–15 and 46–30, respectively (AACC, 2000). For protein
analysis the factor was taken as Nx5,7. The ash content was determined
by ICC Method 104/1 (ICC, 2008).
Zeleny sedimentation value was determined by ICC Standard Method
116/1 (ICC, 2008) and modified Zeleny sedimentation was determined
by incubating 2 h at 37 ◦ C after addition of bromophenol blue according
to Köksel et al. (2000). Wet gluten, dry gluten and gluten index were
determined according to AACC Method 38-12 A by using Glutomatic
System (Perten Instruments AB, Huddinge, Sweden), damaged starch
was determined by AACC Method 76–33 with SD-Matic (Chopin Tech­
nologies, Villeneuve la Garenne, France) and falling number by AACC
Method 56–81 B by using Falling number FN 1900 (Perten Instruments
AB, Kungens Kurva, Sweden) instruments (AACC, 2000).
The farinograph and alveograph parameters were obtained accord­
ing to AACC Method 54–21 and 54-30 A, respectively by using
Farinograph-AT (Brabender GmbH & Co. KG, Duisburg, Germany) and
Alveograph (Chopin, Tripette et Renaud, Villeneuve La Garenne,
France) instruments (AACC, 2000). All the analyses were performed
with three repetitions and the results were given as mean ± standard
deviation.
2.7.2. Bread making procedure
The breads were made according to the AACC Method 10-10 B
(optimized straight dough bread making method) with modifications.
Sucrose, shortening, malt flour and ascorbic acid were not used in the
formulation. The dough was prepared with 100 g of flour (14% moisture
basis), 25 mL yeast solution (8.0%), 25 mL salt solution (6.0%), and
water (according to the farinograph water absorption value). The
amount of amylase was determined as 5-15-25 ppm for the native
amylase (38.6 U/mg) by considering the falling number of the samples.
Regarding the enzyme activity of the commercial alpha-amylase
(FLUKA, Munich, Germany; Aspergillus oryzae, 36.0 U/mg) which was
used as a positive control, the added amount was 5–16-27 ppm. The
ingredients were mixed for 1 min by a pin mixer (National
Manufacturing Co., Lincoln, USA) and approximately 160 g of dough
was obtained. After the first fermentation (30 min, 85% relative hu­
midity) the dough was punched and left for the second fermentation in
the same conditions. Then, the dough was molded, panned and left for
final proof (55 min). The loaves were baked in a rotary oven (Despatch
Oven Co., Minneapolis, USA) at 215 ◦ C for 25 min.
2.7.3. Evaluation of bread characteristics
After cooling the breads at room temperature for 2 h, the volume of
breads were determined by the rapeseed displacement method (AACC
Method 10–05) using a loaf volumeter (National Manufacturing Co.,
Lincoln, USA) (AACC, 2000). The specific volume of breads were
determined by dividing the sample volume (mL) to the sample weight
(g). The color values (L, a, b) of crumb and crust were measured by
HunterLab Miniscan XE Plus (Hunter Associates Laboratory Inc., Reston,
VA, USA). The color difference (ΔE) of breads was calculated according
to the formula below (Mokrzycki & Tatol, 2011). 1 coded parameters
were control breads and 2 coded were α-amylase added bread samples.
The study was performed twice.
3
Food Bioscience 45 (2022) 101492
ΔE
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
( * )2 ( )2
*Lab = L2 − L*1 + (a*2 − a*1 ) + b*2 − b*1
2
2.7.4. Texture profile analysis (TPA)
The breads were cooled at room temperature and stored in poly­
ethylene bags until texture analysis. The TPA of the crumb was deter­
mined by Stable Microsystems TA-XT plus (Godalming, Surrey, England)
according to the modified method of Carr and Tadini (2003) and SMS
(2020) recommendations. The machine was equipped with a 5 kg load
cell and a 36 mm (P/36) aluminum cylindrical probe. The analysis was
performed with two bread slices (stacked together) taken from the
center of each loaf (12.5 mm thickness x2). The test was conducted with
the following settings: pre-test speed 1.0 mm/s, test speed 5.0 mm/s,
post-test speed 5.0 mm/s, strain 40.0%, trigger force auto-20 g, time 5 s.
TPA analysis were repeated 24, 48, 72, 96 and 120 h after baking (days
1, 2, 3, 4, 5 respectively) with three repetitions.
2.8. The statistical analysis
The obtained data were evaluated with the SPSS 25 statistical pro­
gram (SPSS Version 25, 2021, IBM, New York, USA), and the differences
between groups were analyzed by ANOVA analysis of variance analysis.
The differences between groups were determined by Tukey Post Hoc
Analysis in p˂0.95 significance.
3. Results and discussion
3.1. The isolation of fungi
A total of 23 different mold species were isolated from the spring
water of the thermal zones around Afyon, Eskişehir, Uşak and Ankara
provinces. After collection of samples, possible fungi species were pre-
enriched by using SYEM. The presence of amylase enzyme in fungi
were determined by iodine indicator, then enzyme positive samples
were developed by using SYEM (Fig. 1). The amylase activity was
determined by using DNS method and the isolates were identified ac­
cording to morphological, microscopic, and molecular characteristics
(Fig. 2 and Fig. 3). The six isolates (at 40 ◦ C incubation temperature)
belonging to Aspergillus niger in Gazlıgöl (2 species), Aspergillus terreus in
Gazlıgöl (2 species) and Sakarılıca (1 species), and Trichoderma atro­
viride in Sakarılıca (1 species) were found to be high amylase producers.
This is in line with the findings of Costa et al. (2021), indicating
Aspergillus awamori can produce bulk amylase via solid state fermenta­
tion. This is reported to increase ferulic acid, which may improve the
taste properties as well. Moreover, these fungal amylases are reported to
involve in glycoside hydrolase family (Janickova & Janecek, 2020).
3.2. Enzyme production
After deciding the best medium for amylase production, the alpha-
amylase enzyme was produced by the shake flask method from wild
type A. niger, A. terreus, and T. atroviride species. The optimum pH and
temperature parameters for amylase enzyme production were set as
4.5–5.5 pH values and 40–60 ◦ C. These are in line with the findings of
Dahiya et al. (2020) recommending the same temperature interval for
different microbial amylase producer microorganisms. Indeed, the
maximum amylase activity was found for A. niger in Gazlıgöl location as
38.6 U/mg. The amylase activity of Aspergillus terreus and Trichoderma
atroviride were determined as 30.1 and 27.4 U/mg respectively. All
enzymes were powdered by the spray drying method.
3.3. Bread making with enzyme and evaluation of quality
The bread samples were produced with cv. Tosunbey and Kışla, and
about 130 g of bread mass were obtained after cooking. The physical and
A. Ünal et al. Food Bioscience 45 (2022) 101492

Fig. 1. Amylase (+) macroscopic colony appearance of isolates with zone. (A) Aspergillus niger (B) Aspergillus terreus (C) Trichoderma atroviride.

Fig. 2. Under X10 (1) and X40 (2) light microscope, general view and image of conidial (3) heads. (A) Aspergillus niger (B) Aspergillus terreus (C) Tricho­
derma atroviride.

4
A. Ünal et al. Food Bioscience 45 (2022) 101492

high falling number of both cultivars revealed that the amylase activities
were not suitable for bread making and alpha amylase enzyme had to be
added externally.
Hectoliter weight is the weight of 100-L wheat in terms of kg, and it is
affected by grain density, size, shape, and homogeneity. A positive
interaction exists between the flour efficiency of wheat and hectoliter
weight. Thousand kernel weights differ according to the grain density
and size. Since the ratio of endosperm to the other parts of grain is higher
in big and dense grains, the flour efficiency of these grains is accepted as
high (Köksel et al., 2000).
The quality and amount of protein is primarily a characteristic of
wheat species. Environmental factors are particularly effective in the
amount of protein. The quantity and quality of gluten fraction is widely
used in quality assessment. Gluten plays a chief role in viscoelastic
structure related to the kneading, processing, and gas holding capacity
of the bread dough (Elgün & Ertugay, 1992). The sedimentation value is
an important criterion in determining protein quality in wheat. Gluten is
composed of gliadin and glutenin fractions that are insoluble in salt
Fig. 3. DNA gel images as a result of PCR amplification and molecular iden­ water and constitute approximately 85% of the storage proteins. Gluten
tification of molds. forms the skeleton of the dough and helps the formation of bread by
holding the gas produced by the yeast (Elgün et al., 2001).
chemical properties of cv. Tosunbey and Kışla used in bread making The rheological properties of wheat flours were given in Table 3.
have given in Table 2. Hectoliter weight of Tosunbey (76.6 kg/hL), Tosunbey had high-gluten index and the farinograph development time
thousand kernel weight (33.6 g), protein content (12.1%), Zeleny sedi­ was rather high (21.06 min). The stability and the softening degree of
mentation value (45 mL), dry gluten (9.6%) and gluten index (100%) Tosunbey was not recorded since the graph was not indicated a fall
values, flour yield (71%), falling number (348 s) were higher than Kışla. down from 500 consistency level throughout the analysis. It means that
It was determined that the amount of ash (1.49%) and damaged starch the cultivar was stable and the degree of softening was too low. The
(4.8%) content of the Kışla were higher. water absorption value of Kışla was found to be high. Farinograph is an
The amylase activity is substantial in bread production technology instrument used to determine the kneading properties of the dough and
because the sugar content of the medium affects yeast activity during the gives information about the availability of flour for bread making.
fermentation step in bread making. Since no sugar is added to the Testing through farinograph, the water absorption of the flour, the
widespread formulation of bread making in Turkey, the yeast produces rheological properties of the dough during kneading (development time,
gas via using the glucose formed by the degradation of starch by stability, degree of softening) and the dough forming properties of
occurred with enzymatic breakdown. Low amylase activity causes an gluten proteins are determined.
inadequate utilization of sugar by yeast cells, leading to a low bread The farinographic characteristics of flour are mainly related to the
volume. When the enzyme activity is too high, the porous structure of amount and quality of gluten proteins. Flours should have high water
the crumb disrupts, and the bread volume is not at the desired level. absorption, and the kneading time should not be too long for bread
Moreover, the crumb will be sticky. Another parameter; the falling making. If the kneading time is long, it results in the loss of energy and
number gives information about the amylase enzyme activity present in time, so it causes undesired characteristics in the commercial bakery. In
flour. The expected falling number is between 200 and 250 s interval. If contrast, if this time is too short, the quality of flour is getting low. An
the falling number is < 150 s, the amylase activity is very high, and increase in the development time of dough depends on an increase in
>300 s means the amylase activity is low (Köksel et al., 2000). In this protein content. If the development time is long, it means that the dough
study, the falling numbers of the wheat varieties used in bread making, will rise, later on, indicating a long time of kneading and a high amount
Tosunbey and Kışla, were found to be 348 and 317 s, respectively. The and quality of dough.
A short fermentation time lowered the bread volume and disrupted
the porous structure. Stability indicates the durability of dough for
Table 2 processing. The dough is supposed to protect its consistency and should
Physical and chemical properties of wheat cultivars. not soften and increase its water content by no means whatsoever. If the
Tosunbey Kışla stability is short, the processing capacity of the dough will decrease, and
Moisture (%) 10.58 ± 0.03a 10.74 ± 0.01a
Hectoliter weight (kg/hL) 76.60 ± 0.87a 73.63 ± 0.31b Table 3
1000 kernel weight (g) 33.60 ± 0.10a 30.67 ± 0.31b The rheological properties of the dough.
Protein (%)a,b 12.06 ± 0.19a 10.54 ± 0.13b
Ash (%)a 1.43 ± 0.00a 1.49 ± 0.01a Tosunbey Kışla
Grain hardness (%) 28.10 ± 0.00a 21.20 ± 0.00b Farinograph
Flour yield (%) 71.13 ± 0.75a 68.27 ± 0.23b Absorption (%) 56.4 ± 0.05b 57.3 ± 0.24a
Zeleny sedimentation (mL)b 44.67 ± 0.58a 31.33 ± 0.58b Development time (min.) 21.01 ± 0.21b 22.93 ± 0.32b
Modified Zeleny sedimentation (mL)b 53.67 ± 1.53a 32.00 ± 0.00b Stability (min.) 6.87 ± 0.07a 07.58 ± 0.05b
Wet gluten (%)a 28.03 ± 0.42a 27.43 ± 0.40a The degree of softening (BU) 1 63 ± 1.23a 64 ± 1.76a
Dry gluten (%)a 9.57 ± 0.12a 8.60 ± 0.10b Alveograph
Gluten index (%) 99.60 ± 0.00a 84.00 ± 3.50b P (Resistance) (mm) 103.3 ± 5.73A 94.0 ± 4.32B
Falling number (s) 347.67 ± 4.51a 317.33 ± 3.21b L (Extensibility) (mm) 65.0 ± 6.98Aa 49.3 ± 3.86Bb
Damaged starch (%) 4.49 ± 0.03a 4.81 ± 0.03a G (Rising Index) (cm3) 17.9 ± 0.94Aa 15.6 ± 0.65Bb
W (Energy) (10− 4 J) 251 ± 3.86Aa 155 ± 12.81Bb
Values represent mean ± standard deviation for triple determinations; Means in
P/L (Morphological ratio) 1.6 ± 0.26A 1.9 ± 0.19 B
a row with different letters are significantly different (p < 0.05).
2
N x 5.7. Values represent mean ± standard deviation for triple determinations; Means in
a
Given on dry matter. a row with different letters are significantly different (p < 0.05).
b a
It is given on a 14% water basis. BU: Brabender unit.

5
A. Ünal et al. Food Bioscience 45 (2022) 101492

fermentation time is shortened as well. The softening degree is desired to Table 5

be low in qualified wheat used in the bakery (Aydoğan et al., 2012). The crust color values of bread samples.
When the alveograph values of the wheat flours were investigated, Wheat Application L* a* b* ΔE
Tosunbey and Kışla were found to be 251.10− 4 and 155.10− 4 J, Cultivar
respectively. The specific volume values of the loaves of bread made by Tosunbey Control 59.1 ± 7.8 ± 18.3 ± 0.9a
native (5, 15, 25 ppm) and commercial (5, 16, 27 ppm) alpha-amylase 5.04a 0.48a
were presented in Table 4. An increase in alpha-amylase addition YR-5 53.7 ± 7.6 ± 16.6 ± 5.66 ±
leads to a rise in bread volume and specific volume as well. The bread 2.58bc 0.29a 0.34bc 0.09a
YR-15 52.4 ± 7.4 ± 16.6 ± 6.92 ±
volume of Kışla was more than Tosunbey. The isolation of commercial 1.89bc 0.38a 0.53bc 0.11ab
alpha-amylase from A. oryzae and the late inactivation of thermostable YR-25 51 ± 7.2 ± 16.1 ± 8.41 ±
native alpha-amylase at oven temperature was probably affected the 1.63bc 0.32a 0.31b 0.14bc
volume of samples, and higher volume for the native alpha-amylase TIC-5 55.1 ± 7.9 ± 17.1 ± 0.4c 4.18 ±
1.12ac 0.64a 0.08a
samples occurred when compared to the commercial one. The low
TIC-16 52 ± 7.7 ± 16.6 ± 7.30 ±
falling number of Kışla compared to Tosunbey means a high amount of 1.03bc 0.56a 0.28bc 0.12ab
alpha-amylase. The desired falling number in wheat is between 220 and TIC-27 50.6 ± 7.5 ± 16 ± 0.55b 8.81 ±
250 s (Diepenbrock et al., 2005). If enzyme is not added to the flours 1.78b 0.27a 0.17c
with a falling number higher than the average value, a decrease in bread Kışla Control 56.3 ± 7.9 ± 17.6 ±
volume and quality occurs, the dough cannot create enough gas and the 1.97A 0.19A 0.85A
bread crumb is tight (Ünal, 2002). The starch content or quality are the YR-5 52.5 ± 7.4 ± 16.4 ± 4.02 ±
0.66BC 0.46AB 0.58BCD 0.07A
most significant content to generate high quality and high specific vol­
YR-15 50 ± 7.4 ± 15.8 ± 6.57 ±
ume bread (Goesaert et al., 2009). The high damaged starch content of 1.86C 0.42AB 0.31BC 0.06B
the Kışla played a role in high bread volume. The low falling number of YR-25 50.3 ± 7.2 ± 15.7 ± 6.33 ±
Kışla compared to Tosunbey means a high amount of alpha-amylase. 2.47B 0.43B 0.34B 0.07B
According to Olaerts et al. (2018) by removal of 11% and 16% lower TIC-5 55 ± 7.8 ± 17.2 ± 1.36 ±
2.18AC 0.32AB 0.68AD 0.08C
density kernels of mildly and severely sprouted wheat populations, TIC-16 54.3 ± 7.5 ± 17 ± 2.30 ±
respectively, flour enzyme activity decreased and FN and RVA viscosity 1.89AC 0.33AB 0.74AD 0.12AC
readings increased. TIC-27 52.2 ± 7.5 ± 16.8 ± 4.20 ±
The crust color values of bread samples were presented in Table 5. 1.3BC 0.32AB 0.63ACD 0.06A
The crust color can be depending on cooking time, the additive on the Values represent mean ± standard deviation for triple determinations; Means in
surface of bread so it is not possible to compare with the other bread a column with different letters are significantly different (p < 0.05) YR: Native
types. Therefore, the samples including in the study were compared each amylase; TIC: Commercial amylase.
other considering parameters tried. When the alpha-amylase added
samples belong to the Tosunbey were compared with the control, the
lightness (L*) and yellowness (b*) values were decreased, but redness Table 6
(a*) value was increased. The control bread made from Kışla had higher The crumb color values of bread samples.
lightness (L*), redness (a*), and yellowness (b*) values than the amylase Wheat Application L* a* b* ΔE
added. The decrease in b* value was the cause of an increase in glucose Cultivar
amount due to the breakdown of starch by enzyme and accordingly Tosunbey Control 77.1 ± 0.14 ± 14.3 ± –
result in a rise in Maillard reactions. Regarding L* value, a* value was 1.21a 0.06a 0.21a
YR-5 77.6 ± 0.08 ± 14.1 ± 0.54 ±
possibly changed by the effect of Maillard reactions. The color values of
1.65a 0.03b 0.24ac 0.04a
the crumb can be seen in Table 6. It was determined that for the crumb YR-15 76.8 ± 0.06 ± 13.8 ± 0.58 ±
brightness (L*) Tosunbey had higher values than Kışla. In contrast to the 1.41a 0.03b 0.22abc 0.05a
control, the alpha-amylase added bread crumbs had lower a* and b* YR-25 76.6 ± 0.05 ± 13.4 ± 1.03 ±
values. 0.83a 0.03b 0.3b 0.27b
TIC-5 76.6 ± 0.09 ± 14.1 ± 0.54 ±
The textural properties (hardness, cohesiveness, gumminess) of
1.71a 0.04b 0.44ac 0.04a
TIC-16 76.9 ± 0.07 ± 13.8 ± 0.54 ±
1.26a 0.02b 0.25abc 0.04a
Table 4 TIC-27 77.5 ± 0.05 ± 13.6 ± 0.81 ±
The volume and specific volume of bread samples. 1.11a 0.02b 0.51bc 0.24c
Wheat Cultivar Application Bread volume Specific volume Kışla Control 77.2 ± 0.21 ± 14.8 ± –
(mL) (mL/g) 1.38A 0.1A 0.24A
YR-5 77.8 ± 0.08 ± 13.8 ± 1.17 ±
Control 450.7 ± 2.5a 3.4 ± 0.01a 1.87A 0.05B 0.27B 0.24A
YR-5 491.8 ± 14.19bc 3.7 ± 0.12bc YR-15 77.5 ± 0.05 ± 13.7 ± 1.14 ±
YR-15 509.3 ± 10.48cd 3.9 ± 0.08cd 1.92A 0.02B 0.5B 0.21A
Tosunbey YR− 25
520.8 ± 6.65d 4.0 ± 0.06d YR-25 77.6 ± 0.04 ± 13.5 ± 1.37 ±
TIC-5 482.5 ± 8.55b 3.6 ± 0.07b 1.5A 0.02B 0.39B 0.27B
TIC-16 502.2 ± 9.02cd 3.8 ± 0.09bc TIC-5 77.5 ± 0.09 ± 14 ± 0.31B 0.86 ±
TIC-27 507.5 ± 15.33cd 3.9 ± 0.14cd 0.92A 0.03B 0.98C
Control 452.2 ± 3.13A 3.4 ± 0.04A TIC-16 77.5 ± 0.05 ± 13.8 ± 1.05 ±
YR-5 500.5 ± 14.46BC 3.8 ± 0.14BC 0.75A 0.02B 0.39B 0.87A
YR-15 509.7 ± 15.74BC 3.9 ± 0.15C TIC-27 77.4 ± 0.04 ± 13.6 ± 1.23 ±
Kışla YR-25 518.8 ± 13.64C 4.0 ± 0.13C 0.54A 0.01B 0.37B 0.23AB
TIC-5 480.2 ± 2.56D 3.7 ± 0.03B
TIC-16 498.5 ± 5.68BD 3.8 ± 0.04BC Values represent mean ± standard deviation for triple determinations; Means in
TIC-27 510.8 ± 11.65BC 3.9 ± 0.11C a column with different letters are significantly different(p < 0.05)YR: Native
amylase; TIC: Commercial amylase.
Values represent mean ± standard deviation for triple determinations; Means in
a column with different letters are significantly different (p < 0.05) YR: Native
alpha amylase; TIC: Commercial alpha amylase.

6
A. Ünal et al. Food Bioscience 45 (2022) 101492

bread crumbs during 5 days storage were given in Fig. 4. It was deter­ with the Kışla-YR 25 application. The cohesiveness of the alpha amylase
mined that the hardness of the control breads were higher than the alpha added breads were higher than the control breads and during storage the
amylase added breads. Also, increasing alpha amylase levels in breads cohesiveness decreased. Besides, the cohesiveness value of breads made
resulted with decreasing hardness. The lowest hardness was obtained from Tosunbey was higher than that of Kışla. In terms of gumminess the

Fig. 4. Textural properties of breads produced from Tosunbey and Kışla during storage (N: Newton for hardness and gumminess, cohesiveness is unitless, C: Control,
YR-5: Native α-amylase (5 ppm), YR-15: Native α-amylase (15 ppm), YR-25: Native α-amylase (25 ppm), TIC-5: Commercial α-amylase (5 ppm), TIC-16: Commercial
α-amylase (16 ppm), TIC-27: Commercial α-amylase (27 ppm).

7
A. Ünal et al.
control breads had the highest values (Fig. 4).
The gumminess values were inversely proportional to alpha-amylase
level but directly proportional to the storage time. Tosunbey was found
to have a high gumminess compared to Kışla. According to Sarabhai
et al. (2021) addition of enzymes significantly increased specific volume
and crumb springiness while crumb hardness and cohesiveness
decreased in comparison to control in foxtail millet originated bread.
Another study conducted by Barbosa-Rios et al. (2018) supports this
idea, as the incorporation of relatively small amounts of alpha-amylases
and maltogenic amylases improved physical and textural properties of
traditional sweet bread. Staling during storage time decreased as
induced by a better distribution of pores within the bread structure, and
to reduction of water loss due to more stable crust configuration. All
breads were shown in Fig. 5.
4. Conclusion
A total number of 23 fungi were isolated from thermal districts of
Uşak, Afyon, Eskişehir, and Ankara provinces of Turkey. The fungi
species A. niger, Aspergillus terreus, and Trichoderma atroviride were found
as high amylase activity positive species after the macroscopic, micro­
scopic, morphological and molecular identification of amylase producer
Fig. 5. Breads produced from cv. Tosunbey and Kışla by native and commercial α-amylase.
8
Food Bioscience 45 (2022) 101492
isolates. Alpha-amylase was produced by using these native isolates of
fungi via the shake-flask method. The optimum pH and temperature of
fungal amylases were 4.5–5.5 and 40–60 ◦ C. The highest alpha-amylase
activity (38.6 U/mg) was detected in the Aspergillus niger isolate from
Gazlıgöl. The most functional alpha-amylase was obtained in powder
form by spray drying. The effect of native and commercial alpha-
amylase was investigated in different amounts during bread making.
The hardness, one of the crucial indicators of staling, were especially
met in the Kışla variety, showing better results than the control. On the
contrary, the use of encapsulation for maltogenic amylase was found to
increase hardness at the end of fourth day (Haghighat-Kharazi et al.,
2020). The native alpha amylase added breads had higher volume and
specific volume than the commercial alpha amylase added breads. The
volume and color differences between the alpha amylase added breads
and the control were found to be statistically significant (p < 0.05) The
optimum effect for dough processing was observed by adding 5.0 ppm
alpha amylase while the processing and shaping of the dough became
more difficult as the enzyme rate increased. The breads produced with
Kışla were more voluminous and darker than Tosunbey, arising from
lower falling number (318 s) related to higher amylase activity, and the
higher amount of damaged starch. In general, these findings support the
idea of Roth et al. (2019) claimed that; bacterial amylases are well
A. Ünal et al.
applicable to industrial processes due to their high stability, fungal
amylases are recognized as safe and are preferred in the food industry,
even they lack the pH tolerance and stability compared to bacterial ones.
Consequently, when native and the commercial was compared, positive
effects of the native enzyme were observed in bread volume and quality.
Declaration of competing interest
No conflict of interest is present among the authors.
Acknowledgements
The authors would like to thank the General Directorate of Agricul­
tural Research and Policies for supporting the project “Investigation of
Using Potential of Thermal Sourced Native Fungal Alpha Amylase
Enzyme in Improving Bread Quality Using Biotechnological Processes”
numbered with TAGEM/HSGYAD/16/A05/P01/103.
References
AACC. (2000). Approved methods of American association of cereal chemists (10th ed.).
USA: Minnesota.
Adrio, J. L., & Demain, A. L. (2014). Microbial enzymes: Tools for biotechnological
processes. Biomolecules, 4, 117–139.
Amigo, J. M., del Olmo, A., Engelsen, M. M., Lundkvist, H., & Engelsen, S. B. (2021).
Staling of white wheat bread crumb and effect of maltogenic amylases. Part 3:
Spatial evolution of bread staling with time by near infrared hyperspectral imaging.
Food Chemistry, 353, 129–478. https://doi.org/10.1016/j.foodchem.2021.129478,
2021.
Aydoğan, S., Göçmen-Akçacik, A., Şahin, M., Kaya, Y., Koç, H., Görgülü, M. N., &
Ekici, M. (2012). Ekmeklik buğday unlarında alveograf, farinograf ve miksografta
ölçülen reolojik özellikler arasındaki ilişkinin belirlenmesi. Süleyman Demirel
Üniversitesi Ziraat Fakültesi Dergisi, 7, 74–82.
Barbosa-Rios, J. A., Castillon-Jardon, J., Guadarrama-Lezarna, A. Y., Alvarez-Ramirez, J.,
Meraz, M., & Carrillo-Navas, H. (2018). Effect of new generation enzymes addition
on the physical, viscoelastic and textural properties of traditional Mexican sweet
bread. Journal of Cereal Science, 79, 160–167.
Barnett, J., & Hunter, B. (1998). Illustrated genera of imperfect fungi. St. Paul: American
Phytopathological Society Press. ISBN-13: 978-0890541920.
Carr, L. G., & Tadini, C. C. (2003). Influence of yeast and vegetable shortening on
physical and textural parameters of frozen part baked French bread. Lebensmittel-
Wissenschaft und -Technologie- Food Science and Technology, 36, 609–614. https://doi.
org/10.1016/S0023-6438(03)00079-3
Chapman, J., Ismail, A. E., & Dinu, C. Z. (2018). Industrial applications of enzymes:
Recent advances, techniques, and outlooks. Catalysts, 8, 238. https://doi.org/
10.3390/catal8060238
Costa, R. D. S., de Almeida, S. S., Cavalcanti, E. C., Freire, D. M. G., Moura-Nunes, N.,
Monteiro, M., & Perrone, D. (2021). Enzymes produced by solid state fermentation of
agro-industrial by-products release ferulic acid in bioprocessed whole-wheat breads.
Food Research International, 140, 109843.
Dahiya, S., Bajaj, B. K., Kumar, A., Tiwari, S. K., & Singh, B. (2020). A review on
biotechnological potential of multifarious enzymes in bread making. Process
Biochemistry, 99, 290–306. https://doi.org/10.1016/j.procbio.2020.09.002
Diepenbrock, W., Ellmer, F., & Leon, J. (2005). Ackerbau pflanzebauund pflanzenzüchtung.
Stuttgart, Germany: Verlang Eugen Ulmer.
Elgün, A., & Ertugay, Z. (1992). Tahıl İşleme teknolojisi, Erzurum (Vol. 718, p. 376).
Atatürk Üniversitesi Yayınları No.
Elgün, A., Türker, S., & Bilgiçli, N. (2001). Tahıl ve ürünlerinde analitik kalite kontrolü.
Konya: Selçuk üniversitesi ziraat fakültesi, gıda mühendisliği ders notları. Konya
Ticaret Borsası Yayın, 2, 46.
Food Bioscience 45 (2022) 101492
Goesaert, H., Slade, L., Levine, H., & Delcour, J. (2009). Amylases and bread firming – an
integrated view. Journal of Cereal Science, 50, 345–352. https://doi.org/10.1016/j.
jcs.2009.04.010
Haghighat-Kharazi, S., Kasaai, M. R., Milani, J. M., & Khajeh, K. (2020). Antistaling
properties of encapsulated maltogenic amylase in gluten-free bread. Food Sciences
and Nutrition, 8, 5888–5897. https://doi.org/10.1002/fsn3.1865
Hasenekoğlu, I. (1991). Toprak mikrofungusları. Erzurum: Atatürk üniversitesi yayınları
(Vol. 689, p. 30).
ICC. (2008). In International association for cereal science and technology, Vienna, Austria.
ISO 520. (2010). Cereals and pulses- determination of the mass of 1000 grains.
Janickova, Z., & Janecek, S. (2020). Fungal α-amylases from three GH13 subfamilies:
Their sequence-structural features and evolutionary relationships. International
Journal of Biological Macromolecules, 159, 763–772.
Keskin, S.Ö. (2003). Effects of different Ovens and Enzymes on quality Parameters of bread,
master of science thesis. Ankara, Turkey: The Middle East Technical University.
Köksel, H., Sivri, D., Özboy, Ö., Başman, A., & Karaca, H. (2000). Hububat laboratuvarı El
Kitabı (Vol. 47, p. 106). Ankara: Hacettepe Üniversitesi Mühendislik Fakültesi
Yayınları, Yayın.
Leveque, E., Janecek, S., Haye, B., & Belarbi, A. (2000). Thermophilic archaeal
amylolytic enzyme. Enzyme and Microbial Technology, 26, 3–14.
Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing
sugar. Analytical Chemistry, 31, 426–428.
Mobini-Dehkordi, M., & Javan, F. A. (2012). Application of alpha amylase in
biotechnology. CNB Scholar Journals, 1, 39–50.
Mokrzycki, W. S., & Tatol, M. (2011). Colour difference ΔE - a survey. Machine Graphics
and Vision, 20(4), 383–411.
Nguyen, Q. D., Rezessy-Szabo, J. M., Claeyssens, M., Stals, I., & Hoschke, A. (2002).
Purification and characterization of amylolytic enzymes from thermophilic fungus
Thermomyces lanuginosus strain ATCC 34626. Enzyme and Microbial Technology, 3,
345–352.
Olaerts, H., De Bondt, Y., & Courtin, C. M. (2018). Density separation as a strategy to
reduce the enzyme load of preharvest sprouted wheat and enhance its bread making
quality. Food Chemistry, 241, 434–442.
Osho, M. (2018). Industrial Enzyme Technology - Potential Applications. https://doi.org/
10.4018/978-1-5225-5237-6.ch017
Rana, N., Walia, A., & Gaur, A. (2013). Alpha-amylases from microbial sources and its
potential applications in various industries. National Academy Science Letters-India,
36, 9–17.
Rico-Munoz, E., Houbraken, J., & Samson, R. A. (2015). Detection and enumeration of
heat resistant molds. In Y. Salfinger, & M. L. Tortorello (Eds.), Compendium of
methods for the microbiological examination of foods (pp. 45–47). Washington DC:
Apha Press. https://doi.org/10.2105/9780875531755ch2.
Roth, C., Moroz, O. V., Turkenburg, J. P., Blagova, E., Waterman, J., Ariza, A., Ming, L.,
Tianqi, S., Andersen, J., Davies, G. J., & Wilson, K. S. (2019). Structural and
functional characterization of three novel fungal amylases with enhanced stability
and pH tolerance. International Journal of Molecular Sciences, 20, 4902.
Saleem, A., & Mohsen, K. H. E. (2014). Production of amylase by fungi isolated from
legume seeds collected in Almadinah Almunawwarah, Saudi Arabia. Journal of
Taibah University for Science, 8, 90–97.
Sarabhai, S., Tamilselvan, T., & Prabhasankar, P. (2021). Role of enzymes for
improvement in gluten-free foxtail millet bread: It’s effect on quality, textural,
rheological and pasting properties. Lebensmittel Wissenschaft und Technologie, 137,
110365.
Shanmughapriya, S., Kiran, G. S., Selvin, J., Thomas, T. A., & Rani, C. (2010).
Optimization, purification, and characterization of extracellular mesophilic alkaline
cellulase from sponge-associated Marinobacter sp. MSI032. Applied Biochemistry and
Biotechnology, 162, 625–640.
Singh, R., Kumar, V., & Kapoor, V. (2014). Partial purification and characterization of a
heat stable alpha-amylase from a thermophilic Actinobacteria Streptomyces sp.
MSC702. Enzyme Research, 2014, 1–8.
SMS-Stable Micro Systems. (2020). Available in http://www.stablemicrosistems.com.
(Accessed November 2020).
Sundarram, A., & Murthy, T. P. K. (2014). α-Amylase production and applications: A
review. Journal of Applied & Environmental Microbiology, 2, 166–175.
Ünal, S. (2002). Importance of wheat quality and methods in wheat quality determination.
Hububat Ürünleri Teknolojisi Kongre ve Sergisi (pp. 25–37). Turkey: Gaziantep.